In response to Research Objective 10 (Stem Cells, Tissue Repair, and Cell Replacement in Aging) For a number of reasons there has recently been a great increase in interest in neural stem cells, including their significant potential for use in the treatment of a wide range of clinical conditions that involve loss of neural cells. There is also evidence that the cellular and biochemical environment into which such cells are introduced may play a critical role in determining their survival, fate (e.g., migration patterns), and expression of differentiated functions. Proteoglycans and related extracellular matrix glycoproteins with which they interact, such as the tenascins and the Slit proteins, are obvious candidates for molecules that might be expected to exert a critical influence on neural stem cell behavior, yet because of the complex nature of these proteins and the somewhat specialized background and techniques generally associated with their study, virtually no work has yet been directed to this relatively new area of neurobiology. My laboratory has had a longstanding interest in the structures, localization, and functional roles of nervous tissue glycoconjugates. In response to the NIA request for small grant (R03) applications under its Pilot Research Grant Program, and in particular with respect to studies on environmental effects on tissue stem cells and their use in tissue repair and cell replacement in aging (Topic No. 10 in the Announcement), I am proposing studies in the area of my laboratory's expertise that are specifically directed to this end. The aim of these studies is to utilize unique reagents that we have prepared (e.g., antibodies and other probes, purified and well-characterized proteoglycans) to determine qualitatively and semi-quantitatively the production by different types of neural stem cells of a number of chondroitin sulfate and heparan sulfate proteoglycans present in nervous tissue (neurocan, phosphacan, aggrecan, versican, glypican-1) and their extracellular matrix ligands (e.g., hyaluronan, tenascin-C, Slit protein). The neural lineage cells to be investigated in this project will be derived from mouse embryonic stem (ES) cells and P19 embryonal carcinoma cells. We will also begin studies on the effects of proteoglycans and other extracellular matrix macromolecules on neural stem cell proliferation, adhesion, and differentiation. These latter investigations would, if necessary, be completed in the context of an R01 proposal based on the results obtained from the present pilot study, in which they would be extended in directions indicated by our findings. These might include, for example, determination of glycosaminoglycan production by neural stem cells, and possible changes in the composition and localization of proteoglycans as a consequence of neural stem.